Volume 184, number 1 FEBS 2540 May 1985 Further characterization of cardiodigin, zyxwvutsrqponmlkjihgfedcbaZYXWVUTS Na + , K + -ATPase inhibitor extracted from mammalian tissues M. Fagoo and T. Godfraind* Laboratoire de Pharmacodynamie Generale et de Pharmacologic, Universite Catholique de Louvain, UCL 7350, Avenue Emmanuel Mounier 73, B-1200 Bruxelles, Belgium Received 7 February 1985; revised version received 25 March 1985 This study was undertaken to characterize endogenous digitalis-like activity in water extract from mamma- lian tissues. Prepuritied samples obtained from guinea-pig heart were analysed by reverse-phase HPLC using an acetonitrile gradient. The eluent was assayed for its activity as inhibitor of human heart Na+,K+-ATPase and digoxin-like immunoreactivity. Both activities were recovered in the same fraction after two successive chromatographic steps. These results provide further evidence for the presence of an endogenous digitalis- like factor, cardiodigin, in mammalian heart. Cardiodigin Na+,K+-ATPase Reverse-phase HPLC Endogenous Na+,K+-ATPase inhibitor Digoxin radioimmunoassay 1. INTRODUCTION In recent years, a body of experimental evidence has been accumulated that favors the existence of a physiological regulator of the sodium pump. This factor could play an important role in the regulation of sodium excretion [l-3], in the mechanism of action of cardiac glycosides [4-61 and in the pathogeny of hypertension [7-91. We have reported the presence in water extracts of mammalian heart of a factor that cross-reacts with digoxin antibodies and inhibits Na+,K+- ATPase [lo]. This inhibition is specific, reversible, antagonized by potassium and shows species dependency. In addition, the factor displaces specifically bound ouabain. Here, we report that when a guinea-pig heart ex- tract is further purified using two-step reverse- phase HPLC, the peak for inhibition of Na+,K+-ATPase coincides with the peak for specific reactivity with digoxin antibodies. This confirms that the factor so far identified and * To whom correspondence should be addressed previously coined cardiodigin has characteristics analogous to digitalis. 2. MATERIALS AND METHODS 2.1. Digitalis-like factor preparation Guinea-pig hearts were extracted with water by the method in [3], slightly modified. Shortly, after centrifugation and extraction by methanol, the dry residue obtained from 50 g tissue (from about 60 guinea-pigs) was taken up in 10 ml water and extracted 3 times with 10 ml cyclohexane to remove lipids. After lyophilization of the aqueous phase, dry residue equivalent to 6 g original tissue was dissolved in 0.3 ml water and applied to a Bond Elut Cl8 extraction column (Analyticem International). The elution was car- ried out with 3 x 2 ml water and 3 x 1 ml methanol. This last phase was then evaporated at 40°C under low pressure. 2.2. High performance liquid chromatography To purify further the extract, HPLC was per- formed using a Hewlett-Packard HP-1084B 150 Published by Elsevier Science Publishers B. V. (Biomedical Division) OO145793/85/%3.300 1985 Federation of European Biochemical Societies